Transgenic augmentation of erythroferrone in mice ameliorates anemia in adenine-induced chronic kidney disease

Abstract

Anemia is a common and disabling complication of chronic kidney disease (CKD). Current therapies can be burdensome, and full correction of anemia is limited by cardiovascular side effects. New approaches that may offer additional therapeutic options are needed. We explored the anti-anemic effects of erythroferrone, an erythroid hormone that induces iron mobilization by suppressing the master iron-regulatory hormone hepcidin. In a preclinical murine model of adenine-induced CKD, transgenic augmentation of erythroferrone mobilized iron, increased hemoglobin concentrations by approximately 2 g/dl, and modestly improved renal function without affecting systemic or renal inflammation, fibrosis, or markers of mineral metabolism. This study supports the concept that therapeutic augmentation of erythroferrone is a promising approach for alleviating CKD-associated anemia.

Figure 1.. ERFE augmentation ameliorates CKD-associated anemia.

(A) Pilot experimental design: 8-week-old WT-littermate (WT; n = 4) and ERFE-overexpressing transgenic (TG; n = 7) mice were fed 100 ppm iron (Fe) diet with 0.2% adenine for 8 weeks then analyzed. (F) Final experimental design: Mice were weaned at 3 weeks, then WT (n = 7) mice placed on 100 ppm Fe diet and TG (n = 6) placed on 4 ppm Fe diet to prevent iron accumulation prior to the initiation of adenine diet. At 7 weeks of age WT and TG mice were placed on 0.2% adenine diet with 100 ppm Fe for 8 weeks then analyzed. For both experiments: (B, G) blood hemoglobin concentration, (C, H) RBC count, (D, I) mean corpuscular hemoglobin (MCH). (E, J) Mice were weighed weekly. Data are mean ± SEM, analyzed by unpaired-t-test with Welch’s correction (two-tailed). ***P < .001; **P < .01; *P < .05; ns = non-significant.

Figure 2.. ERFE augmentation improves kidney function but does not alter injury markers in adenine-induced CKD.

WT and TG mice from Figure 1F (n=6–7 mice/group) were analyzed after 8 weeks on adenine diet. (A) Body weight, (B) kidney weight, (C) serum creatinine, (D) blood urea nitrogen (BUN). Quantitative PCR (qPCR) analysis of (E) Kim1, (F) Ngal, (G) Krt20 and (H) Slc5a2 expression in kidney tissue. (I, J) Representative H&E-stained kidney sections from WT and TG mice after 8 weeks on adenine diet (10x and 40x magnification, respectively). Data are mean ± SEM, analyzed by unpaired-t-test with Welch’s correction (two-tailed). ***P < .001; **P < .01; *P < .05; ns = non-significant.

Figure 3.. ERFE augmentation lowers hepcidin levels to enhance iron mobilization in adenine-induced CKD.

WT and TG mice from Figure 1F (n=6–7 mice/group) were analyzed. (A) LIC, (B) serum iron, (C-D): tissue iron concentrations in (C) spleen and (D) kidney, (E) serum hepcidin levels, (F) liver hepcidin mRNA expression, and (G) liver hepcidin mRNA/LIC. Data are mean ± SEM, analyzed by unpaired-t-test with Welch’s correction (two-tailed). ****P<.0001, **P < .01; *P < .05; ns = non-significant.

Figure 4.. ERFE augmentation enhances systemic oxygenation without significantly impacting kidney oxygenation in adenine-induced CKD.

WT and TG mice from Figure 1F (n=6–7 mice/group) were analyzed: (A) serum VEGF levels, (B-E): kidney mRNA concentrations by qRT-PCR (B) Vegfa, (C) Gapdh, (D) Angptl1 and (E) Epo. Data are mean ± SEM, analyzed by unpaired-t-test with Welch’s correction (two-tailed). *P < .05; ns = non-significant.

Figure 5.. ERFE augmentation results in unchanged or mildly suppressed kidney BMP signaling in adenine-induced CKD.

WT and TG mice from Figure 1F (n=6–7 mice/group) were analyzed by qRT-PCR of kidney tissue for (A) Id1, (B) Id2, (C) Id4, (D) Smad7, (E) Bmp2 and (F) Bmp7. Data are mean ± SEM, analyzed by unpaired-t-test with Welch’s correction (two-tailed). *P < .05; ns = non-significant.

Figure 6.. ERFE augmentation does not affect kidney fibrosis in adenine-induced CKD.

In WT and TG mice from Figure 1F (n=6–7 mice/group), kidney tissues were analyzed by qRT-PCR for (A) Acta2, (B) Col1a1, (C) Col3a1, (D) Tgfb1 and (E) Fn1. (F) Representative Masson trichrome-stained kidney sections from WT and TG mice after 8 weeks on adenine diet (10x and 40x magnification). Data are mean ± SEM, analyzed by unpaired-t-test with Welch’s correction (two-tailed). ns = non-significant.

Figure 7.. ERFE augmentation does not impact systemic inflammation in adenine-induced CKD.

WT and TG mice from Figure 1F (n=6–7 mice/group) were analyzed by serum 32-plex cytokine/chemokine assay for (A) TNFα, (B) IL-1β, (C) IL-6, (D) IL-1α, (E) IFNγ, and (F) IL-10. Data are mean ± SEM, analyzed by unpaired-t-test with Welch’s correction (two-tailed). ns = non-significant.

Figure 8.. ERFE augmentation does not substantially change mRNA markers of liver or kidney inflammation in adenine-induced CKD.

WT and TG mice from Figure 1F (n=6–7 mice/group) were analyzed by qRT-PCR for (A) Saa1 expression in liver tissue, and (B-D) Tnfa, Il6, and Il1b expression in kidney tissue. Data are mean ± SEM, analyzed by unpaired-t-test with Welch’s correction (two-tailed). **P < .01; ns = non-significant.

Figure 9.. ERFE augmentation does not alter markers of mineral metabolism in adenine-induced CKD.

WT and TG mice from Figure 1F (n=6–7 mice/group) were analyzed. Measured parameters include (A) serum phosphate, (B) serum intact iFGF23, and qPCR analysis of kidney tissue for (C-G) Slc34a1 and Slc34a3 (encoding sodium-dependent phosphate transporters 2A and 2C), Cyp27b1 and Cyp24a1 (encoding the enzymes that respectively generate and break down the active form of vitamin D, 1,25-dihydroxyvitamin D3) and Kl (encoding Klotho, the renal coreceptor for iFGF23). Data are mean ± SEM, analyzed by unpaired-t-test with Welch’s correction (two-tailed). *P < .05; ns = non-significant.